Microtubules are known to play an important role in a variety of intracellular motile processes, including flagellar beating, axoplasmic transport, secretion, and mitosis. The best documented microtubule-associated motile system is the flagellum, in which the dynein (ATPase) arms both bridge adjacent microtubules and serve as motors for flagellar motility. The role of microtubules in axoplasmic transport and secretion is less well understood: electronmicroscopic studies show close associations between organelles and microtubules; drugs which disassemble microtubules either abolish or alter organelle movements. Recent studies have shown that actomyosin as well as microtubules are involved in secretion. The arrangement of microtubules and actomyosin and the mechanisms by which secretory granules are moved within the cytoplasmic cytoskeleton are unknown. The goals of this research are to analyze the structures associated with beta granule movements and to understand the mechanisms responsible for the association of microtubules with membrane-bound organelles. We will a) localize and identify cytoskeletal structures necessary for the transport of beta granules and the secretion of insulin in endocrine pancreas cells, b) reactivate beta granule movements in partially lysed cell models, c) fractionate beta cells and isolate complexes of beta granules attached to microtubules, and d) reassociate beta granules and microtubules in vitro both to determine the proteins responsible for the associations and to reactivate organelle movements along the microtubules. The mechanisms responsible for microtubule-membrane associations will also be studied in eukaryotic cilia, because microtubule-membrane complexes can be isolated in large and pure quantities. High resolution light and electron microscopy will be used to record and analyze beta granule movements and the cytoskeletal elements responsible for the movements. The in vitro associations between microtubules and membranes will be analyzed using both dark field light microscopy and a variety of biochemical techniques.